Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
  • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
  • C12N9/2411—Amylases
  • C12N9/2414—Alpha-amylase (3.2.1.1.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
  • C12N9/2411—Amylases
  • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01001—Alpha-amylase (3.2.1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase

Definitions

• the field relates to animal nutrition and, in particular, to the use of inoculants and enzymes to increase nutrient release in animal diets.
• Microbes can be used to improve the utilization of feed ingredients.
• microbes are widely used as probiotics (also called direct fed microbials) for human health and animal nutrition.
• probiotics also called direct fed microbials
• silage inoculants are additives containing anaerobic lactic acid bacteria that are used to manipulate and enhance fermentation. Benefits include reduced fermentation loss of the silage and enhanced animal performance.
• lactic acid bacteria in commercial inoculants are Lactobacillus plantarum, Enterococcus faecium, various Pediococcus species and other Lactobacillus species.
• Another option is to use enzymes to increase feed digestibility.
• enzymes including phytase, xylanase, beta-glucanase and protease have also been tested for increasing the soluble nutrient levels by pre-incubation with feed components under anaerobic conditions (Ton Nu et al., High-moisture airtight storage of barley and triticale: Effect of moisture level and grain processing on nitrogen and phosphorus solubility. Animal Feed Science and Technology 210 (2015) 125-137).
• Treatment of corn silage with alpha-amylase has also been tested (Leahy et al., Effects of treating corn silage with alpha-amylase and (or) sorbic acid on beef cattle growth and carcass characteristics.
• silage inoculants or enzymes have been helpful in improving nutrient utilization of animal feed, there still is room for improvement. It has been found that a combination of at least one starch hydrolase alone or in combination with at least one protease and at least one inoculant can improve the nutrient utilization of animal feed.
• a method for improving the digestibility of high-moisture grain feed and/or rehydrated grain feed for animals which comprises a) processing the grain feed into grain feed fragments and b) contacting the grain feed fragments of step (a) with at least one starch hydrolase that is stable and active at a pH less than 5.0 in combination with at least one inoculant comprising at least one bacterial strain.
• this starch hydrolase preferably has a starch binding domain that make it capable of hydrolyzing raw starch. Furthermore, the starch hydrolase is selected from the glycoside hydrolase family 13 and/or 15.
• the starch hydrolase is selected from the group consisting of at least one alpha amylase or at least one glucoamylase.
• the method described herein further comprises at least one protease in step (b).
• the protease is an endopeptidase and this endopeptidase is selected from the group consisting of metallopeptidases, serine proteases, threonine proteases and aspartic proteases.
• the at least one inoculant comprises at least one lactobacillus strain.
• the grain feed is selected from the group consisting corn silage, corn grain, barley silage, barley grain, sorghum, sorghum silage, oilseeds or a combination thereof.
• the animal is a ruminant.
• Figure 1 depicts corn kernels broken by using a Buehler Mill.
• Figure 2 depicts the release of soluble nutrient of glucose by the interaction
• Lactobacillus containing inoculant and enzymes from moisture corn Lactobacillus containing inoculant and enzymes from moisture corn.
• the term “about” refers to a range of +/- 0.5 of the numerical value, unless the term is otherwise specifically defined in context.
• the phrase a "pH value of about 6" refers to pH values of from 5.5 to 6.5, unless the pH value is specifically defined otherwise.
• glycoside hydrolase is used interchangeably with “glycosidases”, “glycosyl hydrolases” and “starch hydrolases”
• Glycoside hydrolases assist in the hydrolysis of glycosidic bonds in complex sugar polymers (polysaccharides). Together with glycosyltransferases, glycosidases form the major catalytic machinery for the synthesis and breakage of glycosidic bonds.
• Glycoside hydrolases are classified into EC 3.2.1 as enzymes catalyzing the hydrolysis of 0- or S-glycosides.
• Glycoside hydrolases can also be classified according to the stereochemical outcome of the hydrolysis reaction: thus, they can be classified as either retaining or inverting enzymes.
• Glycoside hydrolases can also be classified as exo or endo acting, dependent upon whether they act at the (usually non-reducing) end or in the middle, respectively, of an oligo/polysaccharide chain. Glycoside hydrolases may also be classified by sequence or structure based methods. They are typically named after the substrate that they act upon.
• starch is used interchangeably with “amylum". It is a polymeric carbohydrate consisting of a large number of glucose units joined by glycosidic bonds and is the most common storage carbohydrate in plants.
• starch can refer to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (CeHioOs wherein X can be any number.
• the term refers to any plant-based material including but not limited to grains, grasses, tubers and roots and more specifically wheat, barley, corn, rye, rice, sorghum, brans, cassava, millet, potato, sweet potato, and tapioca.
• starch binding domain SBD
• CBM carbohydrate binding module
• SBDs can be divided into nine CBM families. As a source of energy, starch is degraded by many various amylolytic enzymes. However, only about 10% of them are capable of binding and degrading raw starch. These enzymes usually possess a distinct sequence-structural module called the starch- binding domain that mediates attachment to starch granules.
• SBD refers to an amino acid sequence that binds preferentially to a starch (polysaccharide) substrate or a maltosaccharide, alpha-, beta and gamma-cyclodextrin and the like. They are usually motifs of approximately 100 amino acid residues found in about 10% of microbial amylolytic enzymes.
• catalytic domain refers to a structural region of a polypeptide which is distinct from the CBM and which contains the active site for substrate hydrolysis.
• granular starch and “raw starch” are used interchangeably herein and refer to raw (uncooked) starch, e.g. , granular starch that has not been subject to gelatinization.
• alpha-amylase is used interchangeably with alpha-1 ,4-D-glucan glucanohydrolase and glycogenase.
• Alpha-amylases (E.C. 3.2.1 .1 ) usually, but not always, need calcium in order to function. These enzymes catalyze the endohydrolysis of alpha-1 , 4-glucosidic linkages in oligosaccharides and polysaccharides.
• Alpha- amylases act on, starch, glycogen, and related polysaccharides and oligosaccharides in a random manner, liberating reducing groups in the alpha-configuration.
• glucoamylase (EC 3.2.1 .3) is used interchangeably with glucan 1 ,4- alpha-glucosidase, amyloglucosidase, gamma-amylase, lysosomal alpha-glucosidase, acid maltase, exo-1 ,4-alpha-glucosidase, glucose amylase, gamma-1 ,4-glucan glucohydrolase, acid maltase, and 1 ,4-alpha-D-glucan hydrolase.
• This enzyme cleaves the last alpha-1 ,4-glycosidic linkages at the non-reducing end of amylose and amylopectin to yield glucose. It also cleaves the alpha-1 ,6-glycosidic linkages.
• protea means a protein or polypeptide domain derived from a microorganism, e.g. , a fungus, bacterium, or from a plant or animal, and that has the ability to catalyze cleavage of peptide bonds at one or more of various positions of a protein backbone (e.g. , E.C. 3.4).
• protease and proteinase
• protes can be used interchangeably. Proteases can be found in animals, plants, fungi, bacteria, archaea and viruses.
• Proteolysis can be achieved by enzymes currently classified into six broad groups based on their catalytic mechanisms: aspartyl proteases, cysteine proteases, trypsin-like serine proteases, threonine proteases, glutamic proteases, and metalloproteases.
• Peptidases can be classified by reaction catalyzed which is a functional classification or by molecular structure and homology which is a MEROPS
• MEROPS classification means a protease having the ability to hydrolyze proteins under acidic conditions.
• Aspartic proteases (EC 3.4.23), also known as aspartyl proteases, an activated water molecule bound to one or more catalytic aspartate residues to hydrolyze a peptide bond in a polypeptide substrate. Generally, they have two highly conserved aspartates in the active site and are optimally active at acidic pH.
• AFP refers to an aspartic fungal protease, that is, an aspartic protease from a fungal organism source.
• metalloprotease is any protease whose catalytic mechanism involves a metal. Most metalloproteases require zinc, but some use cobalt.
• the metal ion is coordinated to the protein via three ligands.
• the ligands coordinating the metal ion can vary with histidine, glutamate, aspartate, lysine, and arginine.
• the fourth coordination position is taken up by a labile water molecule.
• metalloproteinases There are two subgroups of metalloproteinases include (a) exopeptidases, metalloexopeptidases (EC number: 3.4.17), and (b), metalloendopeptidases (3.4.24).
• metalloendopeptidases include ADAM proteins and matrix
• MEROPS database peptidase families are grouped by their catalytic type, the first character representing the catalytic type: A, aspartic; C, cysteine; G, glutamic acid; M, metallo; S, serine; T, threonine; and U, unknown.
• the serine, threonine and cysteine peptidases utilize the amino acid as a nucleophile and form an acyl
• nucleophile is an activated water molecule.
• structural protein fold that characterizes the clan or family may have lost its catalytic activity, yet retain its function in protein recognition and binding.
• Serine protease refers to enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the active site of the enzyme. Serine proteases fall into two broad categories based on their structure: the chymotrypsin-like (trypsin-like) and the subtilisins. In the MEROPS protease classification system, proteases are distributed among 16 superfamilies and numerous families. The family S8 includes the subtilisins and the family S1 includes the
• the subfamily S1 E includes the trypsin-like serine proteases from Streptomyces organisms, such as Streptogricins A, B and C.
• the terms "serine protease”, “trypsin-like serine protease” and “chymotrypsin-like protease” are used interchangeably herein.
• threonine protease refers a family of proteolytic enzymes having a threonine residue within the active site.
• an animal includes all non-ruminant (including humans) and ruminant animals.
• the animal is a non-ruminant animal, such as a horse and a mono-gastric animal.
• mono-gastric animals include, but are not limited to, pigs and swine, such as piglets, growing pigs, sows; poultry such as turkeys, ducks, chicken, broiler chicks, layers; fish such as salmon, trout, tilapia, catfish and carps; and
• the animal is a ruminant animal including, but not limited to, cattle, young calves, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, antelope, pronghorn and nilgai. Ruminants have the unique ability to convert roughage into protein and energy through their microbial/enzyme digestive systems. Accordingly, ruminants play an important role in the earth's ecology and in the food chain.
• ruminants' stomachs have four compartments: the rumen, reticulum, omasum, and abomasum.
• the rumen and the reticulum the food is mixed with saliva and separates into layers of solid and liquid material. Solids clump together to form the cud or bolus.
• Fiber especially cellulose and hemicellulose, is primarily broken down in these chambers by microbes (mostly bacteria, as well as some protozoa, fungi and yeast) into the three major volatile fatty acids (VFAs): acetic acid, propionic acid, and butyric acid.
• VFAs volatile fatty acids
• Protein and nonstructural carbohydrate pectin, sugars, and starches are also fermented.
• the rumen and reticulum have different names, they represent the same functional space as digesta and can move back and forth between them. Together, these chambers are called the reticulorumen.
• the degraded digesta which is now in the lower liquid part of the reticulorumen, then passes into the next chamber, the omasum, where water and many of the inorganic mineral elements are absorbed into the blood stream.
• the digesta is moved to the true stomach, the abomasum.
• the abomasum is the direct equivalent of the monogastric stomach, and digesta is digested here in much the same way.
• Digesta is finally moved into the small intestine, where the digestion and absorption of nutrients occurs.
• Microbes produced in the reticulorumen are also digested in the small intestine. Fermentation continues in the large intestine in the same way as in the reticulorumen.
• Fodder refers to a type of animal feed, is any agricultural foodstuff used specifically to feed domesticated livestock, such as cattle, goats, sheep, horses, chickens and pigs. "Fodder” refers particularly to food given to the animals (including plants cut and carried to them), rather than that which they forage for themselves (called forage). Fodder is also called provender and includes hay, straw, silage, compressed and pelleted feeds, oils and mixed rations, and sprouted grains and legumes (such as bean sprouts, fresh malt, or spent malt). Most animal feed is from plants, but some manufacturers add ingredients to processed feeds that are of animal origin.
• feed is used with reference to products that are fed to animals in the rearing of livestock.
• feed and “animal feed” are used interchangeably.
• grain feed refers to any grain used as feed for domestic livestock, such as cattle, poultry or other animals.
• grain feed refers to the seeds of plants which are typically feed to ruminant animals which may or may not include the outer hull, pod or husk of the seed. Examples include, but are not limited to, barley, corn, oats, sorghum, wheat (triticale), rye, and oilseeds such as soybean and rapeseed.
• high-moisture grain feed refers to grain having at least 23% moisture.
• “high-moisture corn” refers to corn harvested at 23 percent or greater moisture, stored and allowed to ferment in a silo or other storage structure, and used as feed for livestock.
• lage refers to feed preserved by an anaerobic fermentation process
• Endiled refers to plant materials preserved by anaerobic fermentation and typically stored in a bag, bunker or upright silo.
• Oileed refers to any oil-containing seed, nut, kernel, or the like produced by a plant. All such plants, as well as their seeds, nuts, or kernels are contemplated for use herein.
• the oil content of small grains, e.g. , wheat, is only 1 -2%; that of oilseeds ranges from about 20% for soybeans to over 40% for sunflower and rapeseed (canola).
• the major world sources of edible seed oils are soybeans, sunflowers, rapeseed, cotton and peanuts.
• the National Sustainable Agriculture Information Service lists the following as sources of oil for food, specialty, or industrial uses: almonds, apricot kernels, avocado, beech nut, bilberry, black currant, borage, brazil nut, calendula, caraway seed, cashew nut, castor seed, citrus seed, clove, cocoa, coffee, copra (dried coconut), coriander, corn seed, cotton seed, elderberry, evening primrose, grape seed, groundnut, hazelnut, hemp seed, jojoba, linseed, macadamia nut, mace, melon seed, mustard seed, neem seed, niger seed, nutmeg, palm kernel, passion fruit, pecan, pistachio, poppy seed, pumpkin seed, rape seed, raspberry seed, red pepper, rose hip, rubber seed, safflower seed, sea buckthorn, sesame seed, soybean, spurge, stinging nettle, sunflower seed, tropho plant, tomato seed, or walnut.
• “Inoculants” contain bacteria selected to dominate the fermentation of the crops in the silo.
• Silage inoculants are divided in two categories depending on how they ferment a common plant sugar, glucose.
• Homoferm enters produce just lactic acid and include some species of Lactobacillus like Lactobacillus plantarum, Pediococcus species, and Enterococcus species.
• the other category, heterofermenters produce lactic acid, acetic acid or ethanol, and carbon dioxide. Lactobacillus buchneri is the best example of a heteroferm enter.
• the term "functional assay” refers to an assay that provides an indication of a protein's activity.
• the term refers to assay systems in which a protein is analyzed for its ability to function in its usual capacity.
• a functional assay involves determining the effectiveness of the protease to hydrolyze a proteinaceous substrate.
• Enzymes increase digestibility of modern animal feeds, which improve feed: grain ratios for ruminants and monogastric animals alike. Enzymes like cellulase and hemicellulase improve the nutritive value of silage and corn/soy based feeds. Other enzymes like alpha-galactosidase increase the nutritional value of Non-Starch
• NSP Polysaccharides
• a method for improving the digestibility of high-moisture grain feed and/or rehydrated grain feed for animals which comprises a) processing the grain feed into grain feed fragments and b) contacting the grain feed fragments of step (a) with at least one starch hydrolase in combination with at least one inoculant comprising at least one bacterial strain.
• Grain feed can be processed into fragments using any means known to those skilled in the art.
• the grains in most of today's feeds are processed in some manner before being fed. Although some grains can be fed whole, processing, even if it is only grinding, usually makes the nutrients more available to the animal, thus improving digestibility and feed efficiency.
• the primary goal of grain processing is to increase energy (starch) availability to improve animal performance.
• Typical processing methods reduce grain particle size with or without addition of water or steam.
• Some common grain processing methods are steam-flaking, dry-rolling, high-moisture harvesting and storage, and reconstitution (rehydration).
• Commonly used grain processing methods include, but are not limited to, mechanical means such as grinding, cracking, rolling and crimping or thermal processing. Grinding is done using either a hammermill or roller mill. Hammermills grind primarily by the impact of free-swinging hammers on the grain as it falls through the grinding chamber. Screens with specifically sized holes surround the grinding chamber and as the grain particles become small enough, they pass out through the holes. Roller mills have pairs of rolls, often two or three pairs per mill, that crush the grain as it passes between the rolls. The space between rolls can be adjusted to give various particle sizes.
• Exemplary grain feed includes, but is not limited to, corn silage, corn grain, barley silage, barley grain, sorghum, sorghum silage, oilseeds or a combination thereof.
• the starch hydrolase has a starch binding domain wherein said starch hydrolase is capable of hydrolyzing raw starch.
• the starch hydrolase is selected from the glycoside hydrolase family 13 and/or 15.
• the starch hydrolase can be selected from the group consisting of alpha-amylases and glucoamylases.
• a starch binding domain is a structure motif possessed by many starch hydrolases including alpha amylases and glucoamylases (Christiansen et al. , 2009, The carbohydrate-binding module family 20 - diversity, structure, and function, FEBS J. 276: 5006-5029).
• an SBD may also be referred to as a carbohydrate binding module (CBM).
• This structure motif facilitates the hydrolysis of raw starch by the starch hydrolases (Janecek et al. 201 1 , Structural and evolutionary aspects of two families of non-catalytic domains present in starch and glycogen binding proteins from microbes, plants and animals. Enzyme Microb. Technol. 49: 429- 440.
• Glycoside hydrolase family GH13 is the major glycoside hydrolase family acting on substrates containing a-glucoside linkages.
• the a-amylase family represents a clan GH-H of three glycoside hydrolase families GH13, GH70 and GH77.
• the GH13 family includes, but is not limited to a- amylase (EC 3.2.1 .1 ); oligo-1 ,6-glucosidase (EC 3.2.1 .10); a-glucosidase (EC 3.2.1 .20); pullulanase (EC 3.2.1 .41 ); cyclomaltodextrinase (EC 3.2.1 .54); maltotetraose-forming a- amylase (EC 3.2.1 .60); isoamylase (EC 3.2.1 .68); dextran glucosidase (EC 3.2.1 .70); trehalose-6-phosphate hydrolase (EC 3.2.1 .93); maltohexaose-forming a-amylase (EC 3.2.1 .98); maltotriose-forming a-amylase (EC 3.2.1 .
• Glycoside hydrolase family 15 enzymes are exo-acting enzymes that hydrolyze the non-reducing end residues of a-glucosides.
• glucoamylase EC 3.2.1 .3
• amyloglucosidase glucodextranase
• ⁇ , ⁇ -trehalose EC 3.2.1 .28
• fungal glucoamylases present some substrate flexibility and are able to degrade not only a-1 ,4-glycosidic bonds but also a-1 ,6-, a-1 ,3- and a-1 ,2-bonds to a lower degree.
• Acidic stable and active alpha-amylases (EC 3.2.1 .1 ) that can be used are selected from Glycoside Hydrolase Family GH 13. There can be mentioned alpha- amylase from Aspergillus kawachii, A. clavatus. Furthermore, those alpha-amylases having granular starch hydrolyzing activity (GSH) or alpha-amylases that have been recombinantly engineered to have GSH activity can also be used. Such GSH activity is advantageous because these enzymes break down more of the starch, particularly any granular (raw) starch, which may be present in any feed containing molasses and the like.
• GSH granular starch hydrolyzing activity
• Alpha-amylases having GSH activity include, but are not limited to, alpha-amylases obtained from Aspergillus kawachi (e.g. , AkAA), Aspergillus niger (e.g. , AnAA), A.
• TrAA Trichoderma reesei
• Alpha-amylases AkAA, AcAA, and AtAA, have two carbohydrate binding domains, one of which belongs to carbohydrate binding module/domain family 20 (CBM20 or CD20) while the other is sometimes called a secondary binding site (SBS).
• SBSs and CBMs appear to function by 1 ) targeting the enzyme towards its substrate, 2) guiding the substrate into the active site groove, 3) substrate disruption, 4) enhancing processivity, 5) allosteric regulation, 6) passing on reaction products, and/or 7) anchoring to the cell wall of the parent microorganism.
• alpha-amylases that may have GSH activity or enzymes used in carbohydrate hydrolysis processes are commercially available, see, e.g. , TERMAMYL® 120-L, LC and SC SAN SUPER®, SUPRA®, and LIQUEZYME® SC available from Novo Nordisk A/S, FUELZYME® LF from Verenium, and CLARASE® L, SPEZYME® FRED, SPEZYME® XTRA, GC626, and GZYME® G997 available from Danisco, US, Inc., Genencor Division.
• Glucoamylases (EC 3.2.1 .3) are selected from Glycoside Hydrolase Family GH
• glucoamylase 15 include, but are not limited to, glucoamylase from Trichoderma reesei (TrGA and its variant CS4, Brewl ), glucoamylase from Aspergillus fumigatus (AfuGA),
• Proteases also called peptidases or proteinases are enzymes capable of cleaving peptide bonds. Proteases have evolved multiple times, and different classes of proteases can perform the same reaction by completely different catalytic mechanisms. Proteases can be found in animals, plants, bacteria, archaea and viruses.
• Proteolysis can be achieved by enzymes currently classified into six broad groups: aspartic proteases, cysteine proteases, serine proteases, threonine proteases, glutamic proteases, and metalloproteases.
• the method described herein can also include a protease along with the starch hydrolase and inoculant comprising at least one bacterial strain.
• the protease is an endopeptidase selected from the group consisting of metallopeptidases, serine proteases, threonine proteases and aspartic proteases.
• the protease is an acid protease and, more preferably it is an acid fungal protease.
• Any acid proteases can be used in this disclosure.
• acid fungal proteases include those obtained from Aspergillus, Trichoderma, Mucor and Rhizopus, such as A. niger, A. awamori, A. oryzae, Trichoderma reesei, and M. miehei.
• AFP can be derived from heterologous or endogenous protein expression of bacteria, plants and fungi sources.
• a metalloproteinase, or metalloprotease is any protease enzyme whose catalytic mechanism involves a metal. Most metalloproteases require zinc, but some use cobalt. The metal ion is coordinated to the protein via three ligands.
• ADAM proteins Well-known metalloendopeptidases include ADAM proteins and matrix
• Serine proteases are enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the
• proteases active site. They are found ubiquitously in both eukaryotes and prokaryotes. Serine proteases fall into two broad categories based on their structure: chymotrypsin- like (trypsin-like) or subtilisin-like.
• Threonine proteases are a family of proteolytic enzymes harboring a threonine (Thr) residue within the active site.
• Aspartic proteases are a catalytic type of protease enzymes that use an activated water molecule bound to one or more aspartate residues for catalysis of their peptide substrates. In general, they have two highly conserved aspartates in the active site and are optimally active at acidic pH. Nearly all known aspartyl proteases are inhibited by pepstatin
• Silage inoculants are forage additives containing lactic acid producing bacteria (LAB) and other anaerobic bacteria (such as Lactobacillus buchneri). These inoculants are used to manipulate and enhance fermentation in haylage (alfalfa, grass, cereal), corn silage and high-moisture corn. The goals are faster, more efficient fermentation with reduced fermentation losses, improved forage quality and palatability, longer bunk life, and improvements in animal performance. Grain crops that are harvested for silage contain a natural population of both "good” and “bad” microbes. "Good” microbes include lactic acid producing bacteria (LAB) that help ensile the crop. "Bad” or spoilage microbes include Clostridia, enterobacteria, bacilli, yeast and molds that negatively affect silage quality.
• LAB lactic acid producing bacteria
• LAB lactic acid producing bacteria
• Spoilage microbes can cause poor fermentation, excessive dry matter, energy and nutrient losses, development of off flavors/aromas that reduce intakes and can even produce toxins that can compromise the health of animals.
• Silage making relies on the conversion of plant sugars to acid.
• the acid decreases the pH and preserves the forage.
• the first step in the silage making process is to create oxygen-free (anaerobic) conditions through compacting and sealing the forage.
• Anaerobic (oxygen hating) bacteria are present in small numbers on all plant material. Once oxygen-free conditions have been achieved, these bacteria begin to multiply and convert plant sugars to fermentation acids. As fermentation acid levels increase, the pH drops preserving the forage as silage.
• silage There are a variety of naturally occurring bacteria that can be present in silage.
• a lactic fermentation is the most desirable because minimal energy is lost during the fermentation process and lactic acid produces palatable, high feed value silage.
• lactic acid producing bacteria are Lactobacillus plantarum, Enterococcus faecium, various Pediococcus species and other Lactobacillus species.
• Species and specific strains of LAB in commercial inoculants have been selected because they grow rapidly and efficiently, and produce primarily lactic acid. They increase the fermentation rate, causing a more rapid decline in pH, with a slightly lower final pH. The products of fermentation are shifted, resulting in more lactic acid and less acetic acid, ethanol and carbon dioxide. Lactic acid is stronger than acetic acid, and contains almost as much energy as the original sugars.
• Silage inoculants are mostly facultatively anaerobic such as LAB, which means they can grow whether or not oxygen is available. When oxygen is available inoculants help speed up the process of making silage material anaerobic. Once anaerobic conditions are achieved, these same bacteria switch to fast, efficient production of acids (lactic acid and some acetic acid) to reduce pH and prevent growth of spoilage microbes. When oxygen is less available, inoculants limit spoilage microbes that can grow in anaerobic conditions (e.g. Clostridia, listeria).
• Different bacterial strains vary in their ability to produce lactic acid. The most desirable strains are those that can convert sugar to lactic acid with minimal energy and dry matter loss. Any commercially available inoculants can be used. Examples of commercially available inoculants are Pioneer® brand inoculants Pioneer® brand 1 132, 1 127, 1 1 H50 and 1 174. There can also be mentioned Pioneer® brand 1 1 C33 and 1 1 CFT which contain a patented strain of Lactobacillus buchneri which reduces silage heating and spoilage at feed-out time.
• compositions and methods disclosed herein include:
• a method for improving the digestibility of high-moisture grain feed and/or rehydrated grain feed for animals which comprises a) processing the grain feed into grain feed fragments and b) contacting the grain feed fragments of step (a) with at least one starch hydrolase that is both stable and active at a pH less than 5.0 in combination with at least one inoculant comprising at least one bacterial strain.
• starch hydrolase is selected from the group consisting of at least one alpha amylase or at least one glucoamylase.
• step (b) further comprises at least one protease.
• endopeptidase is selected from the group consisting of metallopeptidases, serine proteases, threonine proteases and aspartic proteases.
• Table 1 shows the enzyme type, source organism (when known) and internal or commercial source for samples, and patent references for sequences.
• Table 1 lists the enzyme type, source organism (when known) and internal or commercial source for samples, and patent references for sequences.
• Table 1. List of enzymes, components and biomaterial evaluated.
• TrGA glucoamylase Recombinant Trichoderma Patent US7413879 reseei source
• Pioneer® Brand 1 1 B91 is a high-moisture corn inoculant product designed to: Improve fermentation, retain nutrient content and enhance digestibility of ensiled high-moisture corn. Use in high-moisture corn ensiled at the proper maturity in upright, bunker or bag silos at moistures ranging from 22% to 32%.
• the protein concentration of the enzymes used are given below in Table 2.
• the protein weight ratio of the alpha-amylases, glucoamylases and fungal acidic protease of AkAA/AcAA, TrGA/CS4, and APF are 29%, 70% and 1 %, respectively.
• the Pioneer 1 1 B91 inoculant was prepared by suspending 1 g of the powdery product in 1000g of tap water and mixed well as the diluted inoculant.
• the plastic bags containing the fragmented corn, the inoculant with and without (control) and the enzymes to be tested were vacuum sealed using a vacuum sealer from OBH Nordica. These sealed bags were incubated at 22°C or at -20°C (blank) for 35 days.
• the filtrate (40 ⁇ ) was injected to HPLC analysis on an Aminex HPX-87N HPLC column (Bio-Rad) at a flow rate of 0.6 ml/min, column oven temperature set at 75°C, over 15 min using water as eluent.
• Glucose peak were detected using an inline Rl (refractory index) detector, and the peak areas were integrated using Chromeleon software (Dionex) according to the manufacturer's instructions and compared to the peak areas of glucose standards at 0, 0.025, 0.125, 0.25, 0.5, 1 .0 and 2.0 mg/ml.
• the blank is the pH of the high-moisture corn without an inoculant after 35 days of storage at -20°C instead of 22°C incubation.
• Ctrl is the control in which the high- moisture corn was incubated with the inoculant without the addition of enzymes. For the details of the enzymes added, see Table 2 above. The number (n) of repetitions is 2-5.
• pH4.25 and pH4.27 was measured for the control (inoculant only) and protease plus inoculant treatments respectively.
• Figure 2 and Table 4 show that neither the control sample (inoculant alone) nor the AFP (protease and inoculant) and LAT (alpha amylase and inoculant) treated sample had a glucose amount greater than 0.5 mg per gram corn, in fact the glucose levels in these three treatments were even lower than in the blank due to the
• LAT is a bacterial alpha-amylase that generates maltose, maltosaccharides and glucose. It is believed that glucose production was low due to incubation with the Lactobacillus inoculant that resulted in consumption of some of the glucose generated by LAT.
• the glucose released is in the range 0.1 % to 1 % of the fermented corn.
• the 3-enzyme mixture AcAA+CS4+AFP (alpha amylase, glucoamylase and protease plus inoculant) released a high amount of glucose based on the amount of enzyme dosed (see Table 4).
• the mixture of AkAA+TrGA+AFP generated the next highest amount of glucose based on the amount of enzyme dosed, followed by TrGA, then AfuGA and finally CS4.
• FvGA was found to be less efficient.

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